As a risk factor for arteriosclerosis, oxidized lipoproteins are attracting a lot of attention and extensive studies are underway, and a major antioxidant contained in the lipoprotein is vitamin E (mainly α-tocopherol and γ-tocopherol). Thus, the analysis of vitamin E in each lipoprotein in the blood is thought to be important in understanding the mechanism of arteriosclerosis. Especially, on one of vitamin E, α-tocopherol, abundantly contained in the serum lipoproteins, various academic studies have been reported as follows.
(1) Haidari M. et al., Clin. Chem. 47: 1234 (2001); α-tocopherol/cholesterol levels in LDL are reduced in patients with coronary artery diseases compared to healthy individuals.
(2) Feki M. et al., Clin. Chem. 46: 1401 (2000); α-tocopherol/cholesterol levels in LDL are reduced in patients with coronary artery diseases compared to healthy individuals.
In the blood of patients with diabetes, it is also believed that active oxygen (radical) is abundantly generated and thus oxidant stress becomes enhanced, which attacks the cell causing various complications (Beisswenger P J et al., Diabetes 54: 3274 (2005)). This oxidant stress is one of the causes of vascular inflammation, and is also one of the mechanisms of progress of arteriosclerosis. (Renard C. et al., Diabetes Metab. 32: 15 (2005)). Vitamin E (γ-tocopherol and α-tocopherol) is one of the antioxiding agents of a cell membrane, and is transported via lipoprotein into the body. Vitamin E (γ-tocopherol and α-tocopherol) is also one of the antioxiding agents of the lipoprotein.
Though little is known of the relationship between vitamin E contained in the lipoprotein in the blood and diseases, there are the following reports in addition to the above:
(3) Yolanda B. et al., Arterioscler. Thromb. Vasc. Biol. 17: 127 (1997); α-tocopherol/cholesterol levels in LDL are enhanced in patients with hyperlipidemia compared to healthy individuals.
Little is also known of the relationship between vitamin E in the blood and diabetes or coronary artery diseases; however there are the following reports:
(4) Salonen J T et al., BMJ 31: 1124 (1995); α-tocopherol/cholesterol levels are reduced in patients with diabetes compared to healthy individuals.
(5) Reunanen A. et al., Eur. J. Clin. Nutr. 52(2): 89 (1998); Reduced levels of α-tocopherol lead to a higher risk of developing diabetes.
(6) Sobczak A. et al., J. Chromatogr. 730, 265 (1999); α-tocopherol and γ-tocopherol levels are enhanced in patients with diabetes compared to healthy individuals.
(7) Mayer-Davis E J et al., Diabetes Care 25: 2172 (2002); α-tocopherol levels are reduced in patients with diabetes compared to healthy individuals.
(8) Ohrvall M. et al., J. Intern. Med. 239: 111 (1996); γ-tocopherol levels are reduced in patients with coronary artery diseases compared to healthy individuals but no significant changes in α-tocopherol. The α-tocopherol/γ-tocopherol ratio becomes high.
There are no reports on vitamin E in the lipoproteins other than the above, but since vitamin E are contained in other lipoproteins, research on the etiology and treatment of arteriosclerosis and diabetes are likely to make a great progress by comprehensive investigation on the amount of vitamin E in each lipoprotein.
As a conventional method of analyzing vitamin E in the lipoprotein, a method is known in which after each lipoprotein is separated, vitamin E are extracted by hexane, the extracted solution is dried, and then redissolved in methanol etc., and subjected to a reverse phase chromatography (Haidari M. et al., Clin. Chem. 47: 1234 (2001); Feki M. et al., Clin. Chem. 46: 1401 (2000); Yolanda B. et al., Arteriosclerosis Throbosis and Vascular Biology 17: 127 (1997); Teissier E. et al., Clinical Chemistry 42: 430 (1996)). For the separation of each lipoprotein, there are known a method comprising ultracentrifugation (Haidari M. et al. (2001), supra; Feki M. et al. (2000), supra; Yolanda B. et al. (1997), supra; Teissier E. et al. (1996), supra), a method comprising acrylamide electrophoresis, a method comprising gel filtration chromatography, and a method comprising ion exchange chromatography (Hirowatari Y. et al., J. Lipid Research 44: 1404 (2003); Hirowatari Y. et al., Anal. Biochem. 308: 336 (2002)).
For the separation of each lipoprotein, an ion exchange chromatography has been proposed (Hirowatari Y. et al. (2003), supra) that permits successful separation of each lipoprotein utilizing subtle changes in electric charge by varying the composition of the eluant or the separation conditions. However, the analysis of vitamin E in each lipoprotein requires, as described above, complicated steps of, after separation of each lipoprotein, extracting vitamin E, concentrating to dryness, redissolving, and subjecting to a reverse phase chromatography, and thus it was prone to errors and required a great deal of efforts and time. Though the measurement of changes in vitamin E in each lipoprotein and in the blood and their use as one of the methods for judging the pathological conditions is important in such diseases as diabetes and coronary artery diseases in which oxidative stress and the pathological conditions are closely related, ultracentrifugation is the only available method for measuring vitamin E in each lipoprotein, which is an expensive apparatus and the operation is complicated, and thus research has not made much progress. Furthermore, for vitamin E in the blood extraction by an organic solvent is necessary, the procedure is complicated, and the evaluation result on diabetes is different with researchers.